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Regulating gene expression Goal is controlling Proteins •How many? •Where? •How active? 8 levels (two not shown are mRNA localization & prot degradation) Transcription in Eukaryotes Pol I: only makes 45S-rRNA precursor • 50 % of total RNA synthesis • insensitive to -aminitin •Mg2+ cofactor •Regulated @ initiation frequency RNA Polymerase III makes ribosomal 5S and tRNA (+ some snRNA & scRNA) >100 different kinds of genes ~10% of all RNA synthesis Cofactor = Mn2+ cf Mg2+ sensitive to high [-aminitin] RNA Polymerase II makes mRNA (actually hnRNA), some snRNA and scRNA • ~ 30,000 different gene models • 20-40% of all RNA synthesis • very sensitive to -aminitin Initiation of transcription by Pol II Basal transcription 1) TFIID binds TATAA box 2) TFIIA and TFIIB bind to TFIID/DNA 3) Complex recruits Pol II 4) Still must recruit TFIIE & TFIIH to form initiation complex Initiation of transcription by Pol II Basal transcription 1) Once assemble initiation complex must start Pol II 2) Kinase CTD negative charge gets it started 3) Exchange initiation for elongation factors 4) Continues until hits terminator Initiation of transcription by Pol II Basal transcription 1) Once assemble initiation complex must start Pol II 2) Kinase CTD negative charge gets it started 3) RNA pol II is paused on many promoters! • even of genes that aren’t expressed! •Early elongation is also regulated! Initiation of transcription by Pol II RNA pol II is paused on many promoters! • even of genes that aren’t expressed! (low [mRNA]) •Early elongation is also •regulated! • PTEFb kinases CTD to stimulate processivity & processing Initiation of transcription by Pol II RNA pol II is paused on many promoters! • even of genes that aren’t expressed! (low [mRNA]) •Early elongation is also •regulated! • PTEFb kinases CTD to stimulate processivity & processing • Many genes have short transcripts Initiation of transcription by Pol II RNA pol II is paused on many promoters! • even of genes that aren’t expressed! (low [mRNA]) •Early elongation is also •regulated! • PTEFb kinases CTD to stimulate processivity & processing • Many genes have short transcripts •Yet another new level of control! Transcription Template strand determines next base Positioned by H-bonds until RNA polymerase links 5’ P to 3’ OH in front Transcription Template strand determines next base Positioned by H-bonds until RNA polymerase links 5’ P to 3’ OH in front Energy comes from hydrolysis of 2 Pi Transcription NTP enters E site & rotates into A site Transcription NTP enters E site & rotates into A site Specificity comes from trigger loop Transcription Specificity comes from trigger loop Mobile motif that swings into position & triggers catalysis Transcription Specificity comes from trigger loop Mobile motif that swings into position & triggers catalysis Release of PPi triggers translocation Transcription Proofreading: when it makes a mistake it removes ~ 5 bases & tries again Activated transcription by Pol II Studied by mutating promoters for reporter genes Activated transcription by Pol II Studied by mutating promoters for reporter genes Requires transcription factors and changes in chromatin Activated transcription by Pol II enhancers are sequences 5’ to TATAA transcriptional activators bind them • have distinct DNA binding and activation domains Activated transcription by Pol II enhancers are sequences 5’ to TATAA transcriptional activators bind them • have distinct DNA binding and activation domains • activation domain interacts with mediator • helps assemble initiation complex on TATAA Activated transcription by Pol II enhancers are sequences 5’ to TATAA transcriptional activators bind them • have distinct DNA binding and activation domains • activation domain interacts with mediator • helps assemble initiation complex on TATAA •Recently identified “activating RNA”: bind enhancers & mediator Activated transcription by Pol II •Other lncRNA “promote transcriptional poising” in yeast http://www.plosbiology.org/article/info%3Adoi%2F10.13 71%2Fjournal.pbio.1001715 •lncRNA displaces glucose-responsive repressors & corepressors from genes for galactose catabolism Activated transcription by Pol II •Other lncRNA “promote transcriptional poising” in yeast http://www.plosbiology.org/article/info%3Adoi%2F10.13 71%2Fjournal.pbio.1001715 •lncRNA displaces glucose-responsive repressors & corepressors from genes for galactose catabolism •Speeds induction of GAL genes Euk gene regulation Initiating transcription is 1st & most important control Most genes are condensed only express needed genes not enough room in nucleus to access all genes at same time! must find & decompress gene First “remodel” chromatin: • some proteins reposition nucleosomes • others acetylate histones • Neutralizes +ve charge • makes them release DNA Epigenetics •heritable chromatin modifications are associated with activated & repressed genes Epigenetics ChIP-chip & ChiP-seq data for whole genomes yield complex picture: 17 mods are associated with active genes in CD-4 T cells Generating methylated DNA Si RNA are key: generated from antisense or foldbackRNA Generating methylated DNA Si RNA are from antisense or foldback RNA Primary 24 nt siRNA are generated by DCL3 Generating methylated DNA Si RNA are from antisense or foldback RNA Primary 24 nt siRNA are generated by DCL3: somehow polIV is attracted to make more RNA Generating methylated DNA Si RNA are from antisense or foldback RNA Primary 24 nt siRNA are generated by DCL3: somehow polIV is attracted to make more RNA RDR2 makes bottom strand Generating methylated DNA Si RNA are from antisense or foldback RNA Primary 24 nt siRNA are generated by DCL3: somehow polIV is attracted to make more RNA RDR2 makes bottom strand DCL3 cuts dsRNA into 24nt 2˚ siRNA Generating methylated DNA Si RNA are from antisense or foldback RNA Primary 24 nt siRNA are generated by DCL3: somehow polIV is attracted to make more RNA RDR2 makes bottom strand DCL3 cuts dsRNA into 24nt 2˚ siRNA Amplifies signal!-> extends Methylated region Generating methylated DNA Si RNA are from antisense or foldback RNA Primary 24 nt siRNA are generated by DCL3: somehow polIV is attracted to make more RNA RDR2 makes bottom strand DCL3 cuts dsRNA into 24nt 2˚ siRNA Amplifies signal!-> extends Methylated region These guide “silencing Complex” to target site (includes Cytosine & H3K9 Methyltransferases) mRNA PROCESSING Primary transcript is hnRNA undergoes 3 processing reactions before export to cytosol All three are coordinated with transcription & affect gene expression: enzymes piggy-back on POLII mRNA PROCESSING Primary transcript is hnRNA undergoes 3 processing reactions before export to cytosol 1) Capping addition of 7-methyl G to 5’ end mRNA PROCESSING Primary transcript is hnRNA undergoes 3 processing reactions before export to cytosol 1) Capping addition of 7-methyl G to 5’ end identifies it as mRNA: needed for export & translation mRNA PROCESSING Primary transcript is hnRNA undergoes 3 processing reactions before export to cytosol 1) Capping addition of 7-methyl G to 5’ end identifies it as mRNA: needed for export & translation Catalyzed by CEC attached to POLII mRNA PROCESSING 1) Capping 2) Splicing: removal of introns Evidence: • electron microscopy • sequence alignment Splicing: the spliceosome cycle 1) U1 snRNP (RNA/protein complex) binds 5’ splice site Splicing:The spliceosome cycle 1) U1 snRNP binds 5’ splice site 2) U2 snRNP binds “branchpoint” -> displaces A at branchpoint Splicing:The spliceosome cycle 1) U1 snRNP binds 5’ splice site 2) U2 snRNP binds “branchpoint” -> displaces A at branchpoint 3) U4/U5/U6 complex binds intron displace U1 spliceosome has now assembled Splicing: RNA is cut at 5’ splice site cut end is trans-esterified to branchpoint A Splicing: 5) RNA is cut at 3’ splice site 6) 5’ end of exon 2 is ligated to 3’ end of exon 1 7) everything disassembles -> “lariat intron” is degraded Splicing:The spliceosome cycle Splicing: Some RNAs can self-splice! role of snRNPs is to increase rate! Why splice? Splicing: Why splice? 1) Generate diversity exons often encode protein domains Splicing: Why splice? 1) Generate diversity exons often encode protein domains Introns = larger target for insertions, recombination Why splice? 1) Generate diversity >94% of human genes show alternate splicing Why splice? 1) Generate diversity >94% of human genes show alternate splicing same gene encodes different protein in different tissues Why splice? 1) Generate diversity >94% of human genes show alternate splicing same gene encodes different protein in different tissues Stressed plants use AS to make variant stress-response proteins Why splice? 1) Generate diversity >94% of human genes show alternate splicing same gene encodes different protein in different tissues Stressed plants use AS to make variant Stress-response proteins Splice-regulator proteins control AS: regulated by cell-specific expression and phosphorylation Splicing: Why splice? 1) Generate diversity 2) Modulate gene expression introns affect amount of mRNA produced mRNA Processing: RNA editing Two types: C->U and A->I mRNA Processing: RNA editing Two types: C->U and A->I • Plant mito and cp use C -> U •>300 different editing events have been detected in plant mitochondria: some create start & stop codons mRNA Processing: RNA editing Two types: C->U and A->I • Plant mito and cp use C -> U •>300 different editing events have been detected in plant mitochondria: some create start & stop codons: way to prevent nucleus from stealing genes! mRNA Processing: RNA editing Human intestines edit APOB mRNA C -> U to create a stop codon @ aa 2153 (APOB48) cf full-length APOB100 • APOB48 lacks the CTD LDL receptor binding site mRNA Processing: RNA editing Human intestines edit APOB mRNA C -> U to create a stop codon @ aa 2153 (APOB48) cf full-length APOB100 • APOB48 lacks the CTD LDL receptor binding site • Liver makes APOB100 -> correlates with heart disease mRNA Processing: RNA editing Two types: C->U and A->I • Adenosine de-aminases (ADA) are ubiquitously expressed in mammals • act on dsRNA & convert A to I (read as G) mRNA Processing: RNA editing Two types: C->U and A->I • Adenosine de-aminases (ADA) are ubiquitously expressed in mammals • act on dsRNA & convert A to I (read as G) • misregulation of A-to-I RNA editing has been implicated in epilepsy, amyotrophic lateral sclerosis & depression mRNA Processing: Polyadenylation Addition of 200- 250 As to end of mRNA Why bother? • helps identify as mRNA • required for translation • way to measure age of mRNA ->mRNA s with < 200 As have short half-life mRNA Processing: Polyadenylation Addition of 200- 250 As to end of mRNA Why bother? • helps identify as mRNA • required for translation • way to measure age of mRNA ->mRNA s with < 200 As have short half-life >50% of human mRNAs have alternative polyA sites! mRNA Processing: Polyadenylation >50% of human mRNAs have alternative polyA sites! mRNA Processing: Polyadenylation >50% of human mRNAs have alternative polyA sites! • result : different mRNA, can result in altered export, stability or different proteins mRNA Processing: Polyadenylation >50% of human mRNAs have alternative polyA sites! • result : different mRNA, can result in altered export, stability or different proteins • some thalassemias are due to mis-poly A mRNA Processing: Polyadenylation some thalassemias are due to mis-poly A Influenza shuts down nuclear genes by preventing polyAdenylation (viral protein binds CPSF) mRNA Processing: Polyadenylation 1) CPSF (Cleavage and Polyadenylation Specificity Factor) binds AAUAAA in hnRNA mRNA Processing: Polyadenylation 1) CPSF binds AAUAAA in hnRNA 2) CStF (Cleavage Stimulatory Factor) binds G/U rich sequence 50 bases downstream CFI, CFII bind in between Polyadenylation 1) CPSF binds AAUAAA in hnRNA 2) CStF binds; CFI, CFII bind in between 3) PAP (PolyA polymerase) binds & cleaves 10-35 b 3’ to AAUAAA mRNA Processing: Polyadenylation 3) PAP (PolyA polymerase) binds & cleaves 10-35 b 3’ to AAUAAA 4) PAP adds As slowly, CFI, CFII and CPSF fall off mRNA Processing: Polyadenylation 4) PAP adds As slowly, CFI, CFII and CPSF fall off 5) PABII binds, add As rapidly until 250 Coordination of mRNA processing Splicing and polyadenylation factors bind CTD of RNA Pol II-> mechanism to coordinate the three processes Capping, Splicing and Polyadenylation all start before transcription is done! Export from Nucleus Occurs through nuclear pores anything > 40 kDa needs exportin protein bound to 5’ cap Export from Nucleus In cytoplasm nuclear proteins fall off, new proteins bind • eIF4E/eIF-4F bind cap • also new proteins bind polyA tail • mRNA is ready to be translated!